FIELD OF THE INVENTION
[0001] This invention relates to novel methods for determining the unknown biological activity
of a clostridial neurotoxin in a sample with respect to the known biological activity
of a clostridial neurotoxin in a reference sample. The method comprises the step of
comparing the biological activity of a clostridial neurotoxin preparation with the
biological activity of a standard preparation of a reference clostridial neurotoxin
in certain eumetazoan animals.
BACKGROUND OF THE INVENTION
[0002] In recent years, botulinum neurotoxins have become the standard agent in the treatment
of focal dystonias and spastic indications. Treatment of patients generally involves
injection of the neurotoxin into affected muscle tissue, bringing the agent near the
neuromuscular end plate, i.e. close to the cellular receptor mediating its uptake
into the nerve cell controlling said affected muscle. Various degrees of neurotoxin
spread have been observed. This spread is thought to correlate with the injected amounts
and the particular preparation of neurotoxin injected. Resulting from the spread,
systematic side effects caused by the inhibition of acetylcholine release, may be
observed at nearby muscle tissue. The incidents of unintended paralysis of untreated
muscles can largely be avoided by reducing the injected doses to the therapeutically
relevant level. Overdosing may also be a problem with regard to the patients' immune
system, as the injected neurotoxin may trigger the formation of neutralizing antibodies.
If this occurs, the neurotoxin will be inactivated without being able to relieve the
involuntary muscle activity.
[0003] Differences in the dose equivalents or variations in the determined activity of preparations
such as available sales products or batches produced during the manufacturing process,
commonly a fermentation process, pose an increased risk for patients through possible
side effects and the development of immunity. Therefore, it is of crucial importance
to determine the biological activity of a clostridial neurotoxin contained in said
sales products or production batches reliably (i.e. without significant variation)
and as accurately as possible, in order to adjust the neurotoxin concentration to
a reliable effective dose for the benefit of the patient. This may also serve as an
incentive to the manufacturers to offer formulations allowing optimum exploitation
of biological activity for different therapeutic purposes.
[0004] At present, the botulinum neurotoxin testing is predominantly performed using the
mouse LD
50 assay developed more than 40 year ago (see
Boroff and Fleck, J. Bacteriol. 92 (1966) 1580-1581), which is accepted for potency testing by United States and European regulatory
agencies. This assay involves dosing mice with dilutions of the sample of botulinum
neurotoxin being tested and calculating the dilution at which 50% of the mice would
be expected to die. Since this bioassay requires up to 100 mice for testing a single
sample, and takes up to four days to generate results, there is a large need for alternative
methods that are faster and more accurate, and/or reduce, cause less pain, distress,
and/or replace use of animals such as mice. Any such alternative method, in order
to be acceptable to regulatory agencies for the determination of potency, must be
suitable for the intended purpose of the product in question and must be validated
for sensitivity, specificity, reproducibility and robustness. For botulinum neurotoxin
testing, a suitable potency assay must be used to determine the dose of the final
product or to compare the relative activities of different lots. Because botulinum
neurotoxin activity is dependent upon three functional domains within the protein
molecule, an acceptable potency assay must account for the activity of all domains.
For a discussion of issues relating to the unsolved problem of mouse LD
50 assay replacement, see "
Report on the ICCVAM-NICEATM/ECVAM Scientific Workshop on Alternative Methods to Refine,
Reduce or Replace the Mouse LD50 Assay for Botulinum neurotoxin Testing", NIH Publication
Number 08-6416, February 2008.
[0005] Crisley, Appl Microbiol. 8 (1960) 282-285, describes the development of an toxicity assay for supernatants from frozen stock
cultures of
Clostridium botulinum Type A in goldfish (
Carassius auratus). Despite being known since about 1920, such an assay using goldfish has neither
been used, nor suggested to be used, for determining the potency of clostridial neurotoxin
preparations for pharmaceutical and/or aesthetic use.
[0006] Fenicia et al. (in NIH Publication Number 08-6416,
loc.
cit.) show results from preliminary experiments in microcrustacean
Daphnia magna, demonstrating that
Daphnia magna are sensitive to botulinum neurotoxins, including botulinum neurotoxin serotype A.
[0007] In addition to in vivo assays in animals, several approaches have been pursued to
identifiy assay systems using vertebrate muscular tissue preparations, e.g. comprising
the hind limb muscle or the hind limb extensor digitorum longus muscle of mice and
rats, the plantar muscles of the hind paw e.g. of the mouse, the phrenic nerve-hemidiaphragm
e.g. of the rat or mouse, the levator auris longus muscle e.g. of the mouse and rat,
the frog neuromuscular junction, the biventer cervic muscle of chicks, the rib muscles
or brain tissue e.g. of the mouse and rat or the electric organ of the sea ray.
[0009] The above referenced prior art methods, however, have not resulted in a method certified
by regulatory authorities. Therefore, the out-of-time mouse killing assay must still
be performed, and the need still exists to identify alternative assays.
OBJECTS OF THE INVENTION
[0010] It was an object of the invention to improve the methods of the prior art and to
develop reliable and accurate methods for determining the unknown biological activity
of clostridial neurotoxins in a sample, particularly for pharmaceutical and/or aesthetic
use, so that such methods might be used for regulatory purposes. Such an improved
method would also serve to satisfy the great need for a safe and effective administration.
SUMMARY OF THE INVENTION
[0011] Surprisingly it has been found that the methods for determining the unknown concentration
of botulinum neurotoxins in a sample for clincial and/or aesthetic use can be developed
in non-mammalian species.
[0012] In one aspect, the present invention relates to a method for determining the relative
biological activity of a clostridial neurotoxin preparation, comprising the step of
comparing the biological activity of said clostridial neurotoxin preparation with
the biological activity of a standard preparation of a reference clostridial neurotoxin
in an eumetazoan animal, provided that said animal is not a mammalian species.
[0013] In another aspect, the present invention also relates to an in vivo method for determining
the biological activity of a clostridial neurotoxin preparation, comprising the steps
of (a) contacting an animal with a sample comprising a clostridial neurotoxin preparation;
and (b) comparing the biological effect of the sample with the biological effect of
a reference sample wherein said animal is a Mollusca.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention may be understood more readily by reference to the following
detailed description of the invention and the examples included therein.
[0015] In the context of the present invention, the term "clostridial neurotoxin" refers
to a natural neurotoxin obtainable from bacteria of the class Clostridia, including
Clostridium tetani and
Clostridium botulinum, or to a neurotoxin obtainable from alternative sources, including from recombinant
expression or from genetic or chemical modification. Particularly, the clostridial
neurotoxins have endopeptidase activity. Said neurotoxin may be obtained from a clostridial
cell or by expression of the neurotoxin in a heterologous cell such as
E. coli.
[0016] In the context of the present invention, the term "clostridial neurotoxin preparation"
refers to a composition comprising a clostridial neurotoxin, including, but not limited
to, the raw material obtained from a fermentation process (supernatant, composition
after cell lysis), a fraction comprising a clostridial neurotoxin obtained from separating
the ingredients of such a raw material in a purification process, an isolated and
essentially pure clostridial neurotoxin, and a formulation for pharmaceutical and/or
aesthetic use comprising a clostridial neurotoxin and additionally pharmaceutically
acceptable solvents and/or excipients.
[0017] In the context of the present invention, the term "standard preparation of a reference
clostridial neurotoxin" refers to a preparation comprising a clostridial neurotoxin,
where the biological activity of a given amount of such standard preparation is known.
The biological activity of such standard preparation may, for example, be determined
by the mouse LD
50 assay described above. The LD
50 value defines the quantity of neurotoxin at which 50% of a mouse population is killed
if said quantity is applied to the mice of said mouse population. The LD
50 value defines the so-called "unit" that is the commonly accepted unit used to define
a quantity of a clostridial neurotoxin contained in a sample. The method for determining
said value is known to the person skilled in the art. The method is documented in
the European Pharmacopoeia (6
th edition; available from European Directorate for the Quality of Medicines & Healthcare
(EDQM), 7 allée Kastner, CS 30026, F-67081 Strasbourg, France).
[0018] In the context of the present invention, the term "eumetazoan animal" refers to an
animal belonging to the subkingdom "Eumetazoa" of the Kingdom "Animalia" being characterized
by the presence of tissue. In particular, such animals comprise a nervous system.
The term further includes animals in all development stages that already exhibit tissue
differentiation, including the presence of a nervous system.
[0019] The animals used in the methods of the present invention generally express at least
one receptor for cellular uptake of clostridial neurotoxin (for example, corresponding
to human SV2 for botulinum neurotoxin serotype A), and at least one intracellular
target for such clostridial neurotoxin (for example, corresponding to human SNAP25
for botulinum neurotoxin serotype A). Said target is a protein, which is involved
in the formation of a functional SNARE complex. In certain such embodiments, the animal
comprises at least one gene that is an ortholog of a human gene encoding a receptor
for cellular uptake of such clostridial neurotoxin (for example, human SV2 for botulinum
neurotoxin serotype A), and/or at least one gene that is an orthologue of a human
gene encoding an intracellular target of such clostridial neurotoxin (for example,
human SNAP25 for botulinum neurotoxin serotype A). In one embodiment, said target
is inactivated by interaction with the neurotoxin, in another embodiment, said target
is inactivated by proteolytic cleavage. Alternatively, said animal is a transgenic
animal expressing a suitable target gene from a heterologous source, for example,
a human SNARE protein such as SNAP25 or VAMP. Using a transgenic animal may be necessary
in certain instances, in particular those where the species-specific SNARE proteins
are no substrate for the neurotoxin.
[0020] In one embodiment of the method according to the first aspect of the invention, said
method further comprises the step of administering to said animal, or otherwise exposing
said animal with, at least one sample having a defined amount of said clostridial
neurotoxin preparation.
[0021] In the context of the present invention, the term "administering to said animal ...
at least one sample" refers to a step where such sample is either essentially directly
applied to a part of such animal where the biological activity is supposed to occur
(for example, by intramuscular injection), or is systemically applied to the animal
(for example, by subcutaneous, intraperitoneal or intravenous injection, or by feeding
the animal), and the term "otherwise exposing said animal with ... at least one sample"
refers to any other application, where such animal is brought in contact with such
sample (for example, by adding such sample to the aqueous environment of an animal
living in such an environment).
[0022] In certain embodiments of the method according to the first aspect of the invention,
said animal is an animal belonging to (i) a phylum taken from the list of: Acanthocephala,
Acoelomorpha, Annelida, Arthropoda, Brachiopoda, Bryozoa, Chaetognatha, Cnidaria,
Ctenophora, Cycliophora, Echinodermata, Echiura, Entoprocta, Gastrotricha, Gnathostomulida,
Hemichordata, Kinorhyncha, Loricifera, Micrognathozoa, Mollusca, Nematoda, Nematomorpha,
Nemertea, Onychophora, Orthonectida, Phoronida, Platyhelminthes, Priapulida, Rhombozoa,
Rotifera, Sipuncula, Tardigrada, and Xenoturbellida; (ii) a subphylum taken from the
list of: Tunicata, and Cephalocordata; or (iii) a class taken from the list of: Myxinomorphi,
Petromyzontomorphi, Chondrichthiomorphi, Amphibia, Reptilia, and Aves.
[0023] In another embodiment, said animal is an animal belonging (i) to the phylum Mollusca,;
(ii) to a subphylum taken from the list of: Tunicata and Hexapoda (at all stages of
development, including nymphae, larvae, pupae and imagines), or (iii) to a class taken
from the list of: Amphibia (at all stages of development, including tadpoles and adults)
and Reptilia,
[0024] In another embodiment, said Mollusca is selected from the group consisting of Aplacophora,
Bivalvia, Caudofoveata, Cephalopoda, Gastropoda, Monoplacophora, Polyplacophora and
Scaphopoda.
[0025] In another embodiment, said Bivalvia is selected from the group consisting of Anomalosdesmata,
Cryptodonta, Heterodonta, Paleoheterodonta, Palaeotaxodonta, Pteriomorphia.
[0026] In another embodiment, said Heterodonta is Myoida or Veneroida.
[0027] In another embodiment, said Paleoheterodonta is selected from the group consisting
of Lametilidae, Malletiidae, Neilonellidae, Nuculanidae, Nuculidae, Praenuculidae,
Pristiglomidae, Siliculidae, Tindariidae and Yoldiidae.
[0028] In another embodiment, said said Pteriomorphia is selected from the group consisting
of Arcoida, Limoida, Mytiloida, Ostreoida and Pterioida.
[0029] In certain embodiments of the first aspect of the present invention, the biological
activity is the inhibition of muscle tissue activity.
[0030] In the context of the present invention, the term "inhibition of muscle tissue activity"
refers to the property of clostridial neurotoxins to inhibit activation of muscle
tissue by blocking the release of neurotransmitters from presynaptic vesicles.
[0031] In certain embodiments of the present invention, said inhibition is determined by
directly determining the activity of muscle tissue.
[0032] In the context of the present invention, the term "muscle tissue" refers to any muscle
or part of a muscle the activity of which is accessible for activity measurements,
including both smooth and striated muscles, if available, and individual or multiple
muscle fibers, including whole muscle bundles. The term "directly determining the
activity of said muscle tissue" refers to methods where parameters relating to the
activity of a muscle tissue are directly read out.
[0033] In particular embodiments, said activity is determined by determining one or more
of the following parameters: time to paralysis of said muscle tissue, contraction
rate of said muscle tissue, contraction distance of said muscle tissue, time course
of contraction of said muscle tissue, force of contraction of said muscle tissue,
action potential of said muscle tissue, and time course of the action potential of
said muscle tissue.
[0034] The time to paralysis of muscle tissue may be measured e.g. in seconds or minutes.
According to subvariants, the time to paralysis may be determined based on the muscle
contraction distance (paralysis being achieved once the contraction distance is equal
to 0) or on the muscle twitch frequency (paralysis being achieved once the twitch
frequency is equal to 0). The contraction distance may e.g. be measured in centimeters
or millimeters. The time to paralysis may be defined as the period that passed to
attain half maximum twitch. This is strictly dependent on the neurotoxin concentration.
Methods for determining variation in the contraction rate of the muscle tissue, or
is the variation in the contraction of the muscle tissue, or is the variation in the
force of contraction of the muscle tissue are are known in the art, and are for example
disclosed in
EP 1 597 584 B1. An action potential may, for example, be detected by using electromyography.
[0035] In certain embodiments of the first aspect of the present invention, said inhibition
is determined by indirectly determining the activity of said muscle tissue.
[0036] In the context of the present invention, the term term "indirectly determining the
activity of said muscle tissue" refers to methods involving reading out parameters
resulting from the activity of a muscle tissue.
[0037] In another embodiment, the activity is determined by determining one or more of the
following parameters: LD
50 value for a population of said animal, movement of said animal, time to paralysis
of said animal, muscle-driven change of color in animals with chromatophore cells.
[0038] The determination of LD50 values in an animal according to the methods of the present
invention can be performed in analogy to the mouse LD
50 assay (see
Boroff and Fleck, J. Bacteriol. 92 (1966) 1580-1581). Parameters relating to movement of animals may be, for example, distance, speed
or time-to-paralysis of lateral movement of the animal, or distance (e.g. angle),
frequency, speed of movement or time-to-paralysis of parts of an animal (e.g. of the
tails of tadpoles), in-current and an ex-current siphoning activity of tunicates,
the speed and frequence of color change in Cephalopoda, or the like.
[0039] In certain embodiments of the first aspect of the present invention, the biological
activity is the inhibition of secretory activity.
[0040] In the context of the present invention, the term "inhibition of secretory activity"
refers to the property of clostridial neurotoxins to inhibit activation of cholinergically
controlled secretion, including sweating, lacrimation, glandular secretion, or mucus
production
[0041] In certain embodiments of the present invention, the neurotoxin referred to herein
is a neurotoxin of
C. botulinum and is of serotype A, B, C, D, E, F or G or is a biologically active derivative thereof,
wherein said derivative may be a genetically modified neurotoxin such as a mutant
comprising a deletion of one or more amino acids, an addition of one or more amino
acids and/or a substitution of one or more amino acids. Preferably, said deleted or
added amino acids are consecutive amino acids. According to the teaching of the present
invention, any number of amino acids may be added or deleted, as long as the neurotoxin
is biologically active. For example, 1, 2, 3, 4, 5, up to 10, up to 15, up to 25,
up to 50, up to 100, up to 200, up to 400, up to 500 amino acids or even more amino
acids may be added or deleted. In certain aspects, the present invention refers to
neurotoxins, with an addition of more than 500 amino acids, such as for example up
to 600 or up to 800 additional amino acids, or even more additional amino acids. Accordingly,
a derivative of the neurotoxin may be a biologically active fragment of a naturally
occurring neurotoxin. This neurotoxin fragment may contain an N-terminal, C-terminal
and/or one or more internal deletion(s). "Biologically active", as used in this context,
means, said derivative is taken up into the nerve cell and is capable of denervating
said nerve from the muscle or gland, to which it is connected. Moreover, said clostridial
neurotoxin may be associated with complexing proteins or may be free of complexing
proteins (see below).
[0042] In the context of the present invention, the term "botulinum neurotoxin" refers to
neurotoxins obtainable from
Clostridium botulinum. Currently, seven serologically distinct types, designated serotypes A, B, C, D, E,
F and G, are known, including certain subtypes (e.g. A1, A2, and A3). In
Clostridium botulinum, these neurotoxins are formed as protein complexes comprising a neurotoxic component
and at least another non-toxic protein. Thus, the term "botulinum neurotoxin serotype
A", as used herein, refers to the botulinum neurotoxin complex, such as the 450 kDa
and the 900 kDa complexes which are e.g. obtainable from cultures of
Clostridium botulinum. Preparations comprising such complexes are commercially available, e.g. from Ipsen
Ltd. (Dysport®) or Allergan Inc. (Botox®).
[0043] Moreover, the term botulinum neurotoxin also refers to botulinum neurotoxin which
is free of complexing proteins and which has a molecular weight of approximately 150kDa.
Thus, in the context of the present invention, the term "isolated neurotoxic component
of botulinum neurotoxin serotyp ..." refers to the neuroxic component of the corresponding
botulinum neurotoxin serotype complex free of complexing proteins. This neurotoxic
component is a two-chain polypeptide with a 100 kDa heavy chain joined by a disulfide
bond to a 50 kDa light chain. The heavy chain is responsible for entry into the neuronal
cell, while the light chain comprises an endopeptidase activity responsible for cleaving
one or more proteins that is/are part of the so-called SNARE-complex involved in the
process resulting in the release of neurotransmitter into the synaptic cleft. A high
purity neurotoxic component, free of any other botulinum protein is e.g. available
from Merz Pharmaceuticals GmbH, Frankfurt (Xeomin
®).
[0044] The term "other isolated fragment of any said botulinum neurotoxin .... where such
fragment has neurotoxic activity" refers to a neurotoxin complex that lacks certain
of the non-toxic proteins of a botulinum neurotoxin as defined above, or certain parts
of the neurotoxic component as defined above comprised therein, while maintaining
the neurotoxic activity. Correspondingly, the term "other isolated fragment of any
said ... neurotoxic component, where such fragment has neurotoxic activity" refers
to a neurotoxin component that lacks certain certain parts of the neurotoxic component
of a botulinum neurotoxin as defined above, while maintaining the neurotoxic activity.
Methods for making or identifying such fragments, and methods for identifying whether
such fragments maintain neurotoxic activity, are well known to anyone of ordinary
skill in the art.
[0045] In the context of the present invention, the term "derivative" and the term "variant
or synthethic analogue" refer to a neurotoxin complex or neurotoxic component that
is a chemically, enzymatically or genetically modified derivative of a neurotoxin
as defined herein, including chemically or genetically modified neurotoxin from C.
botulinum. A chemically modified derivative may be one that is modified by pyruvation,
phosphorylation, sulfatation, lipidation, pegylation, glycosylation and/or the chemical
addition of an amino acid or a polypeptide comprising between 2 and about 100 amino
acids . An enzymatically modified derivative is one that is modified by the activity
of enzymes, such as endo- or exoproteolytic enzymes. As pointed out above, a genetically
modified derivative is one that has been modified by deletion or substitution of one
or more amino acids contained in, or by addition of one or more amino acids (including
polypeptides comprising between 2 and about 100 amino acids) to, the proteins of said
neurotoxin or a neurotoxic component thereof. Methods for making such chemically or
genetically modified derivatives, and methods for identifying whether such derivatives
maintain neurotoxic activity, are well known to anyone of ordinary skill in the art.
[0046] In a particular embodiment, said clostridial neurotoxin is selected from the group
of botulinum neurotoxin serotype A, the isolated neurotoxic component of botulinum
neurotoxin serotype A, an isolated fragment of botulinum neurotoxin serotype A or
of the neurotoxic component of botulinum neurotoxin serotype A, where such fragment
has neurotoxic activity, and any variant or synthetic analogue of botulinum neurotoxin
serotype A or of the neurotoxic component thereof, where such variant or analogue
has neurotoxic activity. The term "fragment" relates to a neurotoxin lacking 1, 2,
3, 4, 5, up to 10, up to 15, up to 25, up to 50, up to 100, up to 200, up to 400,
up to 500 amino acids or even more amino acids. Preferably, said amino acids are consecutive
amino acids.
[0047] Particularly, said clostridial neurotoxin is the isolated neurotoxic component of
botulinum neurotoxin of serotype A, which is available from Merz Pharmaceuticals GmbH,
Frankfurt (Xeomin
®).
[0048] In certain embodiments of the first aspect of the present invention, said clostridial
neurotoxin preparation is for pharmaceutical and/or aesthetic use.
[0049] In a particular embodiment of the method according to the first aspect of the invention,
said method comprises the step of comparing the biological activity of a clostridial
neurotoxin preparation for pharmaceutical and/or aesthetic use with the biological
activity of a standard preparation of a reference clostridial neurotoxin in an an
animal belonging (i) to the phylum: Mollusca; (ii) to a subphylum taken from the list
of: Tunicata and Hexapoda (at all stages of development, including nymphae, larvae,
pupae and imagines); or (iii) to a class taken from the list of: Amphibia (at all
stages of development, including tadpoles and adults) and Reptilia, , wherein the
clostridial neurotoxin in said clostridial neurotoxin preparation is selected from
the list of: botulinum neurotoxin serotype A, the isolated neurotoxic component of
botulinum neurotoxin serotype A, any other isolated fragment of botulinum neurotoxin
serotype A or of the neurotoxic component of botulinum neurotoxin serotype A, where
such fragment has neurotoxic activity, and any variant or synthetic analogue of botulinum
neurotoxin serotype A or of the neurotoxic component thereof, where such variant or
analogue has neurotoxic activity.
[0050] In certain embodiments of the present invention, the clostridial neurotoxin in the
clostridial neurotoxin preparation is produced in a fermentation process.
[0052] In particular embodiments, the clostridial neurotoxin preparation is produced in
heterologous cells, i.e. it is produced recombinantly by expressing nucleic acid sequences
encoding a neurotoxin in an appropriate host cells. Methods for the recombinant expression
of clostridial neurotoxin are well known in the art (see, for example,
WO 2006/076902 or
EP 1 206 554). An example of a heterologous cell is
E. coli.
[0053] In certain embodiments of the present invention, the clostridial neurotoxin in said
clostridial neurotoxin preparation and the reference clostridial neurotoxin belong
to the same serotype.
[0054] In particular embodiments, the clostridial neurotoxin in said clostridial neurotoxin
preparation and the reference clostridial neurotoxin are identical.
[0055] In this context, the terms "belong to the same serotype" and "identical" refer to
the nature of the active neurotoxic agent present in the clostridial neurotoxin preparation
and the reference clostridial neurotoxin. For example, when the clostridial neurotoxin
preparation contains the isolated neurotoxic component (i.e. without any complexing
proteins) of botulinum neurotoxin serotype A, and the reference clostridial neurotoxin
is botulinum neurotoxin serotype A (i.e. the full complex), the clostridial neurotoxin
in said clostridial neurotoxin preparation and the reference clostridial neurotoxin
belong to the same serotype, but are not identical. When the clostridial neurotoxin
preparation contains the isolated neurotoxic component (i.e. without any complexing
proteins) of botulinum neurotoxin serotype A, and the reference clostridial neurotoxin
is the isolated neurotoxic component of botulinum neurotoxin serotype A as well, the
clostridial neurotoxin in said clostridial neurotoxin preparation and the reference
clostridial neurotoxin are identical (even though the clostridial neurotoxin preparation
and the preparation containing the reference clostridial neurotoxin may differ in
the amount and concentration of the respective neurotoxin and/or the presence of additional
ingredients such as buffers, excipients etc.).
[0056] In another aspect, the present invention relates to an in vivo method for determining
the biological activity of a clostridial neurotoxin preparation, comprising the steps
of (a) contacting an animal with a sample of the clostridial neurotoxin preparation;
and (b) comparing the biological effect of the sample with the biological effect of
a reference sample; wherein said animal is a Mollusca.
[0057] The term "in vivo" as used herein refers to animals, which are alive. In other words,
the method of the present invention involves contacting a living animal with a sample
and observing the effect that the sample has on the living animal. The term "contacting"
as used herein means exposing the animal or parts of the animal to the sample. One
way of exposing the animal to the sample is to incubate (bathe) the animal in the
sample. To this end, the animal may be kept e.g. in an aquarium, e.g. filled with
sea water (taken from the sea) or in fresh water (taken from a river or lake). The
skilled person will realize that the choice of water depends on the natural environment
of the animal. In order to expose the animal to the sample, a suitable concentration
of the sample may be added to the aquarium. An alternative way to contact the animal
with the sample, is to inject the sample directly into muscular tissue of the animal.
[0058] The skilled person will recognize that a minimal incubation period may be required
to observe an effect of the sample comprising the clostridial neurotoxin on the animal.
Although incubation periods may be different from species to species, the skilled
person can easily establish suitable conditions, which will also depend on the final
concentration of the neurotoxin.
[0059] The term "effect" as used herein refers to activities of the animal. Examples of
such activities are the opening and/or closing of the shell or movement activity of
the animal. In one embodiment, the effect of the sample comprising a clostridial neurotoxin
is determined by an LD
50 assay. In this case, the term "effect" means dying of the animal.
[0060] According to the present invention's teaching, the effect of the sample containing
the clostridial neurotoxin is compared to the effect of a reference sample. The term
"reference sample" refers to a preparation with a known quantity of neurotoxin. In
one embodiment, the effect of the sample is compared to a standard dose-response curve
established by measuring the effect of several reference samples, each containing
a different amount of the reference neurotoxin. In certain embodiments, the quantity
of the reference sample (i.e. the amount per standard unit, e.g. mg/ml, or mg/kg of
body weight) is plotted on the X axis and the response is plotted on the Y axis. Commonly,
it is the logarithm of the quantity that is plotted on the X axis, and in such cases
the dose-response curve is typically sigmoidal, with the steepest portion in the middle,
indicating the half-maximal response. On the Y axis, the response is typically expressed
as percentage of the maximal response observed, so that the quantity causing the half-maximal
response is the quantity causing a 50% response rate, e.g. a 50% lethality (LD
50), 50% inhibition (IC
50), or 50% efficacy (EC
50).
[0061] In one embodiment, the biological effect of the sample and the biological effect
of the reference sample are determined in the same animal species.
[0062] In one embodiment, the biological activity is determined by determining one or more
of the following parameters: time to paralysis of muscle tissue, contraction rate
of muscle tissue, contraction distance of muscle tissue, time course of contraction
of muscle tissue, force of contraction of muscle tissue, action potential of muscle
tissue, and time course of the action potential of muscle tissue.
[0063] In one embodiment, the biological activity is determined by performing an LD
50 for a population of said animal.
[0064] In one embodiment, the animal is a Mollusca selected from the group consisting of
Aplacophora, Bivalvia, Caudofoveata, Cephalopoda, Gastropoda, Monoplacophora, Polyplacophora
and Scaphopoda.
[0065] In one embodiment, the animal is a Bivalvia and the biological activity is determined
by determining the muscle activity involved in closing the Bivalvian shell.
[0066] In one embodiment, the muscle activity is determined by measuring the force required
to open the shell. The force may, for example, be determined by using a force gauge,
such as a digital or mechanical force gauge.
EXAMPLES
Example 1: Determination of LD50 in Blattodea
[0067] A fixed amount of different dilutions from a dilution series of a reference botulinum
neurotoxin serotype A with known biological activity (expressed as mouse units) is
injected into the abdominal part of
Blatella germanica (10 animals for each dilution), and the LD
50 value is determined essentially as well known from the mouse LD
50 assay.
[0068] The same experiment is repeated with a dilution series of the clostridium neurotoxin
preparation in otherwise identical fashion. The amount of clostridium neurotoxin preparation
necessary to achieve LD
50 can then be correlated to the corresponding amounf of reference botulinum neurotoxin
serotype A with known biological activity.
Example 2: Determination of LD50 for Water Animals
[0069] The experiment is performed as described in Example 1, with the exception that water
animals, such as
Aplysia californica, sea squirts, frog tadpoles etc. are used, and that the neurotoxin solutions are
not injected but added to the water reservoir harboring the animals.
Example 3: Determination of IC50 for Water Animals
[0070] The experiment is performed essentially as described in Example 2, with the exception
that non-lethal amounts of the neurotoxin dilutions are being added to the water reservoirs,
and that instead of an LD
50 value an IC
50 value, representing the amount at which 50% of the animals have been paralyzed, or
where a certain movement of the animals or of a part thereof has been paralyzed or
inhibited in 50% of the animals, is determined and used for correlating the biological
activity of the clostridium neurotoxin preparation with the known biological activity
of the reference botulinum neurotoxin serotype A.
Example 4: Determination of Inhibition of Muscle Activity in Mytilis Edulis
[0071] In mussels of the class "Bivalvia", the two-part shells are held tightly together
by an adductor muscle. Methods and apparatuses for determining the forces required
for opening the mussels have been described in the literature (see, for example,
Lavoie, Biol Bull 111 (1956) 114-122). Similar types of experiments as described in Lavoie can be used for deterimining
the inhibition of muscle activity by clostridial neurotoxins.
[0072] In a first experiment, a number of
Mytilis edulis, each having a length of 5 cm ± 0.25 cm, are kept in an experimental setting as shown
in
El-Shenawy et al. ( Pak. J. Applied Sci. 3 (2003) 687-702), and the valve movement and the maximum opening of the valves in fresh seawater
is recorded. Next, each mussel is attached to an apparatus as shown in Lavoi, exerting
a constant force by attaching a weight with a mass of 800 g. Mussels that open by
more than 1 mm are discarded. Next, a dilution series of a reference botulinum neurotoxin
serotype A with known biological activity (expressed as mouse units) is added to the
aqueous environment of the mussels, and the concentration of reference botulinum neurotoxin
is determined that results in 50% of the mussels having a gap of more than 90% of
the average maximum opening . Such a concentration of the reference botulinum neurotoxin
is then the LD
50 value (if the mussels are killed so that the shells open), or an IC
50 value (if the mussels survive, but the adductor muscle activity is inhibited).
[0073] By performing the same experiment using a dilutions series of the clostridium neurotoxin
preparation in otherwise identical fashion, the biological activity of the clostridium
neurotoxin preparation can be correlated with the known biological activity of the
reference botulinum neurotoxin serotype A.
[0074] Alternatively, the same assay format can be used in order to measure the force required
to open the shells of
Mytilis edulis up to more than 90% of the average maximum opening , when a given amount of the reference
botulinum neurotoxin serotype A is added to the test system. By using a series of
amount of the reference botulinum neurotoxin serotype A, a dose-response (i.e. inhibition)
curve can be established, and a linear range can be identified. Then, one or more
samples of the clostridium neurotoxin preparation to be tested can be added to the
assay system, and the respective forces required to open the shells of
Mytilis edulis up to more than 90% of the average maximum opening can be determined. By correlating
the forces required with the dose-response curve for the reference botulinum neurotoxin
serotype A, the biological activity of the clostridium neurotoxin preparation can
be determined with respect to the known biological activity of the reference botulinum
neurotoxin serotype A.
Example 5: Determination of Inhibition of Color Change in Cephalopoda
[0075] Cephalopoda are able to achieve rapid changes of the colors of their skin as a means
of camouflage. The color change is brought about by chromatophore organs, which are
cytoelastic sacs of pigment with radial muscles. These muscles are innervated by motoneurons.
Under physiological conditions, color change is a rapid process. For example, the
color change in
Octopus vulgaris resulting from getting in contact with a diver was determined to be completed in
about 2 seconds (see
Hanlon, Current Biology 17 (2007) 400-R404).
[0076] For the assay a Cephalopoda such as
Octopus vulgaris or
Sepia officinalis is placed in an aquarium. By applying an external stimulus, such as the appearance
of a silhouette simulating a predatory enemy for the Cephalopoda, or by placing the
aquarium on a monitor surface and changing the color of the picture displayed by the
monitor, the Cephalopoda is caused to perform its color change, and the time for achieving
such changes under physiological conditions is determined.
[0077] Next, the experiment is repeated several times using increasing (non-lethal) amounts
of a reference botulinum neurotoxin serotype A being added to the water in the aquarium.
Preferably, separate experiments are performed for each data point. The corresponding
times for achieving the color changes in the presence of botulinum toxin are monitored.
Preferably, a concentration range is identified where a linear relationship between
the amounts being added and the corresponding times for achieving the color change
exists.
[0078] Finally, another animal in a separate aquarium is treated with a defined amount of
the clostridium neurotoxin preparation to be tested (preferably a botulinum neurotoxin
serotype A as well, most preferably of the same type as the reference botulinum neurotoxin
serotype A), and the time for achieving the color change in the presence of the clostridium
neurotoxin preparation is monitored. By comparing that time with the set of data of
the experiments with the reference botulinum neurotoxin serotype A or the extrapolation
therefrom, the biological activity of the clostridium neurotoxin preparation, expressed
in terms of the known biological activity of the reference, is possible.